The present invention is directed to inhibiting alcohol effects on cell adhesion, and more particularly to method and compound for antagonizing inhibition effects of alcohol on cell adhesion, and further to the use of alcohol inhibition antagonists in prophylaxis or treatment of toxic effects of alcohol.
Alcoholism is a major public health problem in the United States, costing the nation approximately $167 billion annually as a result of medical expenses, accidents, alcohol-related violence, and lost productivity. Two common neurological disorders associated with drinking are fetal alcohol syndrome (FAS) and memory disorders. FAS is a common and preventable cause of mental retardation that results from heavy maternal drinking during pregnancy. FAS affects about one in 1,000 U.S. infants and about 6 percent of the offspring of alcoholic mothers. Children with FAS exhibit mental retardation, growth retardation, malformations of the brain, face, and heart, and behavioral disorders, while children with less severe fetal alcohol effects exhibit neurobehavioral deficits. In addition, alcohol abuse can lead to neurological disorders in adults, disrupting memory and learning.
The brain malformations result in part from a failure of neurons (nerve cells) to migrate correctly from their place of origin to their destination during brain development. Neuronal migration depends heavily on the actions of cell adhesion molecules, which are chemical tags that protrude from nerve cell membranes and stick to like molecules on adjacent cells or other molecules in the extracellular space. Cell adhesion molecules guide neuronal migration during the formation of the nervous system, and their disruption results in brain malformations.
L1 is a cell adhesion molecule that plays a critical role in brain development. Children with mutations in the gene for L1 have brain malformations similar to those seen in children with FAS. Previously, we found that ethanol inhibits the adhesiveness of some cells that have been engineered to express human L1, suggesting that ethanol""s effects on this molecule might contribute to FAS. L1 may also play an important role in learning and memory in the mature nervous system. The finding was striking because of the similarity in brain lesions between children with mutations in the gene for L1 and children with fetal alcohol syndrome. Cell adhesion molecules, such as L1, influence neuronal migration and the extension and bundling of neuronal processes, functions that are essential for normal human nervous system development. L1 is also believed to play a role in long-term synaptic changes that may influence learning and memory.
Ethanol is known to cause serious injury to both the developing and mature nervous system (Reference 1). Recent evidence suggests that alcohols alter nervous system function by interacting directly with selective neural proteins, including ion channels, kinases, and transporters (References 2 and 3). Experiments with the homologous series of 1-alcohols reveal different cutoffs for alcohol effects on diverse native and purified proteins (References 4-6). For alcohols below the cutoff, potency increases as a function of increasing hydrophobicity; alcohols above the cutoff are less potent or inactive. The inactivity of 1-alcohols of greater hydrophobicity, than those below the cutoff, has been taken as evidence that the active 1-alcohols interact with protein rather than lipid sites. The size of the alcohol cutoff for the GABAA (a receptor for the neurotransmitter GABA) and glycine receptors can be manipulated by substituting single amino acids within the transmembrane region of a protein subunit (Reference 7), indicating a striking degree of target specificity. Diverse alcohol targets appear to comprise a hydrophobic crevice that binds methyl groups and a hydrophilic allosteric site that interacts with the hydroxyl group (Reference 8).
L1 is an immunoglobulin cell adhesion molecule that regulates neuronal migration, axon fasciculation (bundling of nerve processes), and growth cone guidance (leading edge of actively extending nerve processes), through homophilic and heterophilic interactions (Reference 9). We have shown that clinically-relevant concentrations of ethanol inhibit cell-cell adhesion mediated by L1 in transfected fibroblasts and in the NG108-15 neuroblastomaxc3x97glioma cell line (derived from rat and mouse tumors) (References 10-13). In NG108-15 cells, ethanol also inhibits morphogenetic changes induced by BMP-7 (growth factor that induces expression of L1 in neural cells), a powerful inducer of L1 and N-CAM (cell adhesion molecule different from L1) gene expression (Reference 10). Because of the similarity in brain lesions in children with fetal alcohol syndrome and those with mutations in the gene for L1, we have speculated that ethanol effects on L1 could play a role in the pathophysiology of fetal alcohol syndrome (Reference 11). Interestingly, ethanol potently inhibits L1-mediated neurite extension in cerebellar granule cells (Reference 14).
1-alcohol inhibition of cell-cell adhesion demonstrates an abrupt cutoff effect between 1-butanol and 1-pentanol (References 10-11), consistent with a direct effect on L1 or an associated protein.
U.S. Pat. Nos. 5,496,851; 6,169,071; and 6,169,072, are directed to method and compounds for modulating or inhibiting cell-cell adhesion.
Although increasing evidence suggests that alcohols act within specific binding pockets of selective neural proteins, however, antagonists at these sites have not yet been identified.
The present invention is based on the discovery that 1-alcohols from methanol through 1-butanol inhibit with increasing potency the cell-cell adhesion mediated by the immunoglobulin cell adhesion molecule L1. The inventors surprisingly found that an abrupt cutoff exists after 1-butanol, with 1-pentanol and higher 1-alcohols showing no effect, and that strict structural requirements must be met for alcohol inhibition of cell-cell adhesion in L1-transfected NIH/3T3 (a rat fibroblast cell line) and in NG108-15 neuroblastomaxc3x97glioma hybrid cells treated with BMP-7, an inducer of L1 and N-CAM. The target site discriminates the tertiary structure of straight-chain and branched-chain alcohols and appears to comprise both a hydrophobic binding site and an adjacent hydrophilic allosteric site. Modifications to the 2- and 3-carbon positions of 1-butanol increased potency, whereas modifications that restrict movement about the 4-carbon abolished activity. The effects of ethanol and 1-butanol on cell-cell adhesion were antagonized by 1-pentanol (1C50=715 xcexcM) and 1-octanol (IC50=3.6 xcexcM). Antagonism by 1-octanol was complete, reversible, and non-competitive. 1-octanol also antagonized ethanol inhibition of BMP-7 morphogensis in NG108-15 cells. 1-Octanol and related compounds are believed to prove useful in determining the role of altered cell adhesion in ethanol-induced injury of the nervous system.
The principal object of the present invention is to provide a method and compound for antagonizing inhibition effects of alcohol on cell adhesion. This is based on the discovery that straight-chain and branched-chain alcohols have highly specific structural requirements for inhibition of cell-cell adhesion. We discovered that 1-pentanol and 1-octanol completely abolish the effects of ethanol and 1-butanol on cell-cell adhesion, and that the effects of ethanol are on the morphogenetic actions of BMP-7.
Another object of the present invention is to provide a method and compound for the prophylaxis or treatment of neurotoxic effects of alcohol, particularly beverage alcohol, i.e., ethanol.
An additional object of the present invention is to provide a method and compound for the prophylaxis or treatment of fetal alcohol syndrome, memory disorders, malformations of the brain, cognitive learning disorders, neuro behavioral disorders, neurological disorders, teratogenesis, and alcohol-related memory disorder and alcohol addiction in adults.
In accordance with the present invention, a method of antagonizing inhibition effects of alcohol on cell adhesion, includes contacting a cell-adhesion molecule expressing cell with an effective amount of a compound, wherein the compound comprises an alcohol with five or more carbons. More particularly, the compound comprises 1-pentanol, 1-octanol, and structural derivatives thereof.